Type i-e crispr-cas systems for eukaryotic genome editing

Abstract

Compositions and methods are provided for modification of a target sequence in the genome of a cell. The methods and compositions employ a cascade system and guide polynucleotide to provide an effective system for targeting, binding to, and modifying or altering target sequences within the genome of a cell or organism. Also provided are systems further comprising an endonuclease. Compositions and methods are also provided for guide polynucleotide/endonuclease systems comprising at least one nuclease covalently or non-covalently linked to, or assembled with, at least one protein subunit of a cascade, and for compositions and methods for direct delivery of endonucleases.

We claim: 
     
         1 . A method for modifying a polynucleotide sequence of a target site in the genome of a plant cell, comprising providing to the plant cell the following molecules:
 (a) a Cascade comprising at least one component selected from the group consisting of: Cse1, Cse2, Cas5, Cash, and Cas7;   (b) a molecule capable of nicking, cleaving, or editing at least one nucleotide of the polynucleotide sequence of the target site; and   (c) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to the polynucleotide target sequence in the genome of said eukaryotic cell; and   wherein the guide polynucleotide and Cascade form a complex capable of recognizing and binding to the polynucleotide sequence of the target site; and wherein the polynucleotide sequence of the target site is modified by the addition at least one nucleotide, the substitution of at least one nucleotide, the deletion of at least one nucleotide, the chemical alteration of at least one nucleotide, or any combination of the preceding.   
     
     
         2 . The method of  claim 1 , wherein the molecule of (b) is a Cas3 nuclease. 
     
     
         3 . The method of  claim 1 , wherein the molecule of (b) is Fok1. 
     
     
         4 . The method of  claim 1 , wherein the molecule of (b) is I-TevI. 
     
     
         5 . The method of  claim 1 , wherein the molecule of (b) is an engineered Cas3 nickase. 
     
     
         6 . The method of  claim 5 , wherein nicking occurs on both strands of a double-stranded polynucleotide to create a double-strand break. 
     
     
         7 . The method of  claim 1 , wherein at least one of the components of (a) is a fusion protein further comprising a transcriptional activator. 
     
     
         8 . The method of  claim 1 , wherein a plurality of components of (a) each further comprise a transcriptional activator. 
     
     
         9 . The method of  claim 1 , wherein the molecule of (b) is a nuclease domain that is fused to at least one of the components of (a). 
     
     
         10 . The method of  claim 9 , wherein the nuclease domain is I-TevI. 
     
     
         11 . The method of  claim 9 , wherein the nuclease domain is Fok1. 
     
     
         12 . The method of  claim 1 , wherein the target sequence is an endogenous gene of the cell. 
     
     
         13 . The method of  claim 1 , wherein the target sequence is heterologous to the cell. 
     
     
         14 . The method of  claim 1 , further comprising providing to the cell a heterologous polynucleotide. 
     
     
         15 . The method of  claim 14 , wherein the heterologous polynucleotide is a DNA repair template, and wherein the polynucleotide sequence of the target site is modified to include at least one base pair alteration as compared to its native state. 
     
     
         16 . The method of  claim 14 , wherein the heterologous polynucleotide is a donor DNA molecule, and wherein the polynucleotide sequence of the target site is modified by the incorporation of the heterologous polynucleotide. 
     
     
         17 . The method of  claim 1 , wherein at least one component of the Cascade in (a) is provided as a polynucleotide sequence in a recombinant construct encoding the polypeptide component. 
     
     
         18 . The method of  claim 1 , wherein at least one component of the Cascade in (a) is provided as a polypeptide. 
     
     
         19 . The method of  claim 1 , wherein the components of the Cascade in (a) and the molecule of (b) are provided as polynucleotides in one or more recombinant constructs. 
     
     
         20 . The method of  claim 1 , wherein the guide polynucleotide of (c) is provided as a DNA molecule that is operably linked to a heterologous regulatory element. 
     
     
         21 . The method of  claim 20 , wherein the heterologous regulatory element is a polII promoter or a polIII promoter. 
     
     
         22 . The method of  claim 1 , wherein the guide polynucleotide of (c) is provided as a polyribonucleotide molecule, optionally further comprising at least one deoxyribonucleotide. 
     
     
         23 . The method of  claim 1 , wherein at least one of the molecules provided to the cell is provided via  Agrobacterium -mediated transformation. 
     
     
         24 . The method of  claim 1 , wherein at least one of the molecules provided to the cell is provided via particle bombardment. 
     
     
         25 . The method of  claim 1 , wherein the modification of the polynucleotide sequence of the target site results in transcriptional activation of a gene. 
     
     
         26 . The method of  claim 1 , further comprising placing the plant cell in a medium that promotes viability, and screening the cell for the presence or absence of a trait of interest. 
     
     
         27 . The method of  claim 1 , further comprising placing the plant cell in a medium that promotes growth, generating a plant from the plant cell, and screening the plant for the presence or absence of a trait of agronomic interest. 
     
     
         28 . The method of  claim 1 , wherein the plant cell is obtained or derived from a plant selected from the group consisting of: corn ( Zea mays ), rice ( Oryza sativa ), rye ( Secale cereale ), sorghum ( Sorghum bicolor, Sorghum vulgare ), millet (e.g., pearl millet ( Pennisetum glaucum ), proso millet ( Panicum miliaceum ), foxtail millet ( Setaria italica ), finger millet ( Eleusine coracana )), wheat ( Triticum  species, for example  Triticum aestivum, Triticum monococcum ), sugarcane ( Saccharum  spp.), oats ( Avena ), barley ( Hordeum ), switchgrass ( Panicum virgatum ), pineapple ( Ananas comosus ), banana ( Musa  spp.), palm, ornamentals, turfgrass, other grass, soybean ( Glycine max ),  Brassica  species (for example but not limited to: oilseed rape or Canola) ( Brassica napus, B. campestris, Brassica rapa, Brassica juncea ), alfalfa ( Medicago sativa ), tobacco ( Nicotiana tabacum ),  Arabidopsis  ( Arabidopsis thaliana ), sunflower ( Helianthus annuus ), cotton ( Gossypium arboreum, Gossypium barbadense ), and peanut ( Arachis hypogaea ), tomato ( Solanum lycopersicum ), and potato ( Solanum tuberosum ). 
     
     
         29 . A synthetic composition comprising:
 (a) a plant cell;   (b) Cse1, Cse2, Cas5, Cash, Cas7;   (c) a molecule capable of nicking or cleaving a polynucleotide sequence of a target site in the plant cell; and   (d) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a polynucleotide target sequence in the plant cell.   
     
     
         30 . The method of  claim 29 , wherein the molecule of 29(c) is a Cas3 nuclease, 
     
     
         31 . The method of  claim 29 , wherein the molecule of 29(c) is Fok1. 
     
     
         32 . The method of  claim 29 , wherein the molecule of 29(c) is I-TevI. 
     
     
         33 . The method of  claim 29 , wherein the molecule of 29(c) is an engineered Cas3 nickase. 
     
     
         34 . The method of  claim 29 , wherein at least one of the components of 29(b) is a fusion protein further comprising a transcriptional activator. 
     
     
         35 . The method of  claim 29 , wherein a plurality of components of 29(b) each further comprise a transcriptional activator. 
     
     
         36 . The method of  claim 29 , wherein the molecule of 29(c) is a nuclease domain that is fused to at least one of the components of 29(b). 
     
     
         37 . The synthetic composition of  claim 29 , wherein at least one of the members of 29(b) further comprises a heterologous nuclease domain or a transcriptional activator. 
     
     
         38 . The method of  claim 29  further comprising a heterologous polynucleotide. 
     
     
         39 . The method of  claim 38 , wherein the heterologous polynucleotide is a DNA repair template, and wherein the polynucleotide sequence of the target site is modified to include at least one base pair alteration as compared to its native state. 
     
     
         40 . The method of  claim 38 , wherein the heterologous polynucleotide is a donor DNA molecule, and wherein the polynucleotide sequence of the target site is modified by the incorporation of the heterologous polynucleotide. 
     
     
         41 . The synthetic composition of  claim 29 , wherein the plant cell is obtained or derived from a plant selected from the group consisting of: corn ( Zea mays ), rice ( Oryza sativa ), rye ( Secale cereale ), sorghum ( Sorghum bicolor, Sorghum vulgare ), millet (e.g., pearl millet ( Pennisetum glaucum ), proso millet ( Panicum miliaceum ), foxtail millet ( Setaria italica ), finger millet ( Eleusine coracana )), wheat ( Triticum  species, for example  Triticum aestivum, Triticum monococcum ), sugarcane ( Saccharum  spp.), oats ( Avena ), barley ( Hordeum ), switchgrass ( Panicum virgatum ), pineapple ( Ananas comosus ), banana ( Musa  spp.), palm, ornamentals, turfgrasses, other grass, soybean ( Glycine max ),  Brassica  species (for example but not limited to: oilseed rape or Canola) ( Brassica napus, B. campestris, Brassica rapa, Brassica juncea ), alfalfa ( Medicago sativa ), tobacco ( Nicotiana tabacum ),  Arabidopsis  ( Arabidopsis thaliana ), sunflower ( Helianthus annuus ), cotton ( Gossypium arboreum, Gossypium barbadense ), and peanut ( Arachis hypogaea ), tomato ( Solanum lycopersicum ), and potato ( Solanum tuberosum ). 
     
     
         42 . A method for modifying a polynucleotide sequence of a target site in the genome of a plant cell, comprising providing to the plant cell the following molecules:
 (a) a Cascade comprising Cse1, Cse2, Cas5, Cas6, and Cas7;   (b) a Cas3 nickase; and   (c) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to said polynucleotide target sequence in the genome of said plant cell; and   
       wherein the guide polynucleotide and Cascade form a complex capable of recognizing and binding to the polynucleotide sequence of the target site; and wherein the polynucleotide sequence of the target site is modified by the addition at least one nucleotide, the substitution of at least one nucleotide, the deletion of at least one nucleotide, the chemical alteration of at least one nucleotide, or any combination of the preceding. 
     
     
         43 . A method for modifying a polynucleotide sequence of a target site in the genome of a plant cell, comprising providing to the plant cell the following molecules:
 (d) a Cascade comprising Cse1, Cse2, Cas5, Cas6, and Cas7;   (e) I-TeVI; and   (f) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to said polynucleotide target sequence in the genome of said plant cell; and   
       wherein the guide polynucleotide and Cascade form a complex capable of recognizing and binding to the polynucleotide sequence of the target site; and wherein the polynucleotide sequence of the target site is modified by the addition at least one nucleotide, the substitution of at least one nucleotide, the deletion of at least one nucleotide, the chemical alteration of at least one nucleotide, or any combination of the preceding. 
     
     
         44 . A method for modifying a polynucleotide sequence of a target site in the genome of a plant cell, comprising providing to the plant cell the following molecules:
 (g) Cascade genes cse1, cse2, cas5, cas6, and cas7;   (h) a gene encoding a Cas3 nickase; and   (i) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to said polynucleotide target sequence in the genome of said plant cell; and   
       wherein the guide polynucleotide and Cascade form a complex capable of recognizing and binding to the polynucleotide sequence of the target site; and wherein the polynucleotide sequence of the target site is modified by the addition at least one nucleotide, the substitution of at least one nucleotide, the deletion of at least one nucleotide, the chemical alteration of at least one nucleotide, or any combination of the preceding. 
     
     
         45 . A method for modifying a polynucleotide sequence of a target site in the genome of a plant cell, comprising providing to the plant cell the following molecules:
 Cascade genes cse1, cse2, cas5, cas6, and cas7;   (k) a gene encoding I-TevI; and   (l) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to said polynucleotide target sequence in the genome of said plant cell; and   
       wherein the guide polynucleotide and Cascade form a complex capable of recognizing and binding to the polynucleotide sequence of the target site; and wherein the polynucleotide sequence of the target site is modified by the addition at least one nucleotide, the substitution of at least one nucleotide, the deletion of at least one nucleotide, the chemical alteration of at least one nucleotide, or any combination of the preceding.